Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS FOR REGULATING BETA-OXIDATION IN PLANTS
FIELD OF THE INVENTION
The invention relates to the genetic manipulation of plants, particularly to
altering lipid metabolism in plants and plant seeds.
BACKGROUND OF THE INVENTION
Vegetable oil, particularly oil extracted from seeds, is an important
agricultural commodity. Currently, most of the vegetable oil that is produced
is
directly or indirectly consumed by humans. Because the oxidation of vegetable
oils
can lead to undesirable odors and flavors in the oil that consumers fmd
unpalatable,
agricultural scientists initiated efforts to improve the oxidative stability
of vegetable
oils. The scientists have significantly improved this aspect of oil quality.
The
oxidative stability of the vegetable oil is primarily related to the number of
double
bonds in its fatty acids. That is, fatty acids with several double bonds are
known to be
more unstable than fatty acids with fewer double bonds. Thus, scientists have
worked
to improve shelf life and oxidative stability by reducing the amount of the
trienoic
fatty acid, a-linolenic acid.
Other work has concentrated on producing oils with specific fatty acid
compositions. Recently, medical science has provided evidence that the
replacement
of fats in the human diet with oils rich in the monounsaturated fatty acid,
oleic acid, is
beneficial to human cardiovascular health. Medical and nutritional experts
alike are
now advocating the replacement of fats in the human diet with oils rich in the
monounsaturated fats. As a result of the increased demand for oils rich in
monounsaturated fatty acids, agricultural scientists have concentrated their
efforts on
developing new plant varieties for the production of vegetable oils that are
rich in
oleic acid. High-oleic canola (Brassica spp.), safflower, and sunflower oils
are now
available.
More recently, agricultural scientists have initiated efforts to genetically
engineer crop plants to produce seed oils containing unusual fatty acids, such
as
epoxy and hydroxy fatty acids. Primarily, scientists have initiated these
efforts to
produce oil containing such fatty acids for use in industrial applications.
Such fatty
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acids find use as plasticizers, lubricants, surfactants, components of paints
and
renewable raw materials in a myriad of industrial syntheses. One goal of the
scientists is to develop renewable replacements for the non-renewable,
petroleum-
based, raw materials upon which industry currently depends. Thus, in the
future,
vegetable oils with unusual fatty acids are likely to increase in industrial
importance
as the world's finite petroleum reserves diminish.
While some progress has been made in the genetic engineering of crop plants
for the production of seed oils with unusual fatty acids, agricultural
scientists have
experienced difficulties in achieving levels of these fatty acids that make
the industrial
use of the oil economically practical. The challenge remains for agricultural
scientists
to increase the levels of the these fatty acids in the seeds of crop plants
used for oil
production.
SUMMARY OF THE INVENTION
Compositions and methods are provided for decreasing (3-oxidation in plants.
The compositions comprise nucleotide sequences encoding acyl-CoA
thioesterases,
particularly maize peroxisomal acyl-CoA thioesterases. The compositions find
use in
methods for modulating acyl-CoA levels in a plant. The compositions and
methods
find use in increasing the level of oil in a plant or part thereof by reducing
the rate of
fatty acid catabolism, particularly (3-oxidation, in peroxisomes. The methods
involve
modulating the level of an acyl-CoA thioesterase in a plant, particularly in
peroxisomes. By increasing or decreasing the level of the acyl-CoA
thioesterase in
the peroxisome, the level of CoASH is altered, causing a decline in (3-
oxidation.
Methods for optimizing a plant for seed oil production are also provided.
Such methods involve increasing or decreasing the level of an acyl-CoA
thioesterase
in plant peroxisomes and also involve decreasing the level or activity of one
or more
additional proteins in a plant which affect (3-oxidation either directly or
indirectly.
Such additional proteins may be enzymes involved directly in J3-oxidation or
proteins
that control peroxisome biogenesis and abundance.
Transformed plants, plant tissues and plant cells, and seeds thereof are
provided. Additionally provided are isolated proteins comprising acyl-CoA
thioesterases.
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DETAILED DESCRIPTION OF THE INVENTION
The invention is drawn to compositions and methods for altering lipid
metabolism in plants. Particularly, the invention provides compositions and
methods
for decreasing (3-oxidation in plants and plant seeds. By decreasing (3-
oxidation in
plant tissues that accumulate oil, such as, for example, developing seeds and
fruits,
increased accumulation' of oil or constituents of the oil can be achieved in a
plant or
part thereof. Thus, the compositions and methods of the invention find use in
increasing in a plant, or part thereof, the accumulation of oil and/or the
level of
particular constituents of the oil. The compositions and methods find further
use in
increasing the accumulation of unusual fatty acids, or triacylglycerols
comprising at
least one unusual aryl chain, in plants.
The invention provides compositions comprising isolated nucleotide
molecules which comprise nucleotide sequences encoding acyl-CoA thioesterases,
particularly maize acyl-CoA thioesterases, more particularly maize peroxisomal
acyl-
CoA thioesterases. The invention further provides expression cassettes
comprising
such nucleotide sequences and isolated proteins encoded by such nucleotide
sequences.
Acyl-CoA thioesterase catalyzes the hydrolysis of an acyl-CoA to a free fatty
acid and CoASH. Acyl CoA-thioesterases are found in the cytosol, mitochondria,
and
peroxisomes in yeast and animal cells (Smith (1994) FASEB J8:1248-1259).
Recently, acyl-CoA thioesterase has been implicated in controlling fatty acid
(3-
oxidation in fungi. In a strain of yeast possessing a deletion mutation of the
gene
encoding a peroxisomal acyl-CoA thioesterase, growth of the strain on a medium
containing fatty acids is impaired relative to the growth of a wild-type
strain on the
same medium. (Jones et al. (1999) J. Biol. Chem. 274:9216-9223).
Interestingly,
overexpression of the peroxisomal acyl-CoA thioesterase gene can also lead to
reduced (3-oxidation. Recently, Chang et al. ((1999) J. Cell Sci. 112:1579-
1590)
demonstrated that in human skin fibroblasts which had been genetically
manipulated
to overexpress peroxisomal aryl-CoA thioesterase had a reduced peroxisome
abundance. Because the peroxisomes are one of the sites of (3-oxidation in
human
cells, a decrease in peroxisome abundance in such cells is likely to be
correlated with
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a decrease in (3-oxidation. The combined results from yeast and human skin
fibroblasts indicate that an optimal level of acyl-CoA thioestexase is
essential to
maintaining a normal rate of (3-oxidation in a cell. The combined results also
indicate
that abnormally high or low levels of acyl-CoA thioesterase can perturb
metabolic
processes or peroxisome abundance, resulting in a decreased flux of carbon
through
the (3-oxidation pathway.
Methods are provided for modulating acyl-CoA thioesterase expression in a
plant. The methods find use in modulating acyl-CoA thioesterase expression in
a
plant or part thereof. By "modulating acyl-CoA thioesterase expression" is
intended
decreasing or increasing the level of acyl-CoA transcripts, protein, enzyme
activity or
combination thereof. The methods of the invention employ the aryl-CoA
thioesterase
nucleotide sequences of the invention. The methods involve transforming at
least one
cell of a plant with at least a portion of: a nucleotide sequence set forth in
SEQ ID
NO: 1, a nucleotide sequence comprising at least 45% identity to the
nucleotide set
I S forth in SEQ ID NO: l, or a nucleotide sequence encoding the amino acid
sequence
set forth in SEQ ID NO: 2.
The methods of the invention do not depend on a particular method of
transforming a plant or cell thereof with a nucleotide construct. Any method
for
transforming a plant with a nucleotide construct known in the art may be
employed in
the methods of the invention including stable, transient and virus-mediated
transformation methods. If expression of the acyl-CoA thioesterase nucleotide
sequence is desired in a plant, the nucleotide construct may additionally
comprise a
promoter that drives expression in a plant cell. Such a promoter is operably
linked to
the nucleotide sequence of the invention for the production of either sense or
antisense transcripts. Preferred methods of the invention additionally involve
regenerating the transformed cell into a transformed plant.
Promoters of interest are seed-preferred, embryo-preferred, constitutive,
chemically regulatable, tissue-preferred, and developmentally regulated
promoters.
Preferred promoters of the invention are seed-preferred and embryo-preferred
promoters.
If desired, the level of thioesterase transcripts, or protein or enzyme
activity
may be assessed to determine if the desired change in acyl-CoA thioesterase
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expression has been achieved. Methods for assessing transcripts, proteins and
acyl-
CoA thioesterase are known in the art. Transcripts may be assessed by, for
example,
northern blot analysis or RNase protection assays, and acyl CoA thioesterase
protein
may be determined by, for example, western blotting. Acyl-CoA thioesterase
activity
may be determined by enzyme activity assays.
Methods are provided for decreasing (3-oxidation in a plant. Such methods
find use in increasing the level of oil or the level of a constituent of the
oil in a plant
or part thereof. Generally, plants tissues do not accumulate substantial
levels of oil.
However, certain plant species have tissues, particularly in seeds and fruits,
that
accumulate substantial amounts of oil. Preferred plant parts or tissues of the
invention
are seeds, embryos and fruits. In seeds that produce oil, the oil is primarily
synthesized during seed development. As oil synthesis proceeds in the
developing
seed, the predominant pathway for lipid degradation in plants, (3-oxidation,
is also
operable. Thus, the accumulation of oil within a developing seed can be
decreased
because of (3-oxidation. Reduced carbon in the form of fatty acids and their
derivatives can be shunted from oil synthesis to (3-oxidation, resulting in a
reduced
accumulation of oil. The present invention provides methods that can increase
the
level of oil, or at least one constituent of the oil, synthesized in a plant
or part thereof.
Such methods involve decreasing (3-oxidation by modulating aryl-CoA
thioesterase
expression in a plant.
While the invention does not depend on any particular biological mechanism,
decreasing acyl-CoA thioesterase expression may reduce (3-oxidation by
reducing the
rate of production of CoASH. Because a fatty acid must be esterified with
CoASH to
form fatty acyl-CoA before (3-oxidation, limiting the formation of CoASH will
reduce
fatty acyl-CoA production, and thus decrease [3-oxidation. Alternatively,
increasing
or overexpressiiig acyl-CoA thioesterase is known to reduce peroxisome
abundance.
Because the peroxisome a major site of (3-oxidation in plant tissues, a
decrease in
peroxisome abundance in a cell is likely to decrease the flux of carbon to j3-
oxidation.
The methods for decreasing [3-oxidation involve modulating acyl-CoA
thioesterase expression, particularly peroxisomal acyl-CoA thioesterase
expression.
The methods do not depend on a particular method of modulating acyl-CoA
thioesterase expression in a plant. Any method known in the art for modulating
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expression may be employed singly or in combination to achieve the desired
result. It
is recognized that acyl-CoA thioesterase expression in a plant may be affected
by
genetically manipulating a plant to increase or decrease the level of acyl-CoA
thioesterase protein produced. For example, an increase in acyl-CoA
thioesterase
protein may be achieved by transforming the plant with a nucleotide construct
comprising a promoter that drives expression in a plant cell operably linked
to a
nucleotide sequence encoding ~an acyl-CoA thioesterase. Alternatively, acyl-
CoA
thioesterase expression may be affected by modifying the kinetic properties of
an
endogenous acyl-CoA thioesterase through site-directed alterations of the
coding
sequence of the endogenous gene resulting in changes in the amino acid
sequence of
the encoded enzyme. Such site-directed alterations may be accomplished by any
method known in the art including, but not limited to, a chimeraplasty-based
method
involving a nucleotide construct of the invention. In yet another approach,
acyl-CoA
thioesterase expression may be decreased or increased by administering to a
plant
allosteric effectors or enzyme activity inhibitors of the thioesterase.
Preferably, such
effectors or inhibitors are administered to a plant before or during oil
synthesis in the
plant. Additionally, modulating the level in a cell of one or more specific
proteins,
other than or in addition to, an acyl-CoA thioesterase, can alter acyl-CoA
thioesterase
in the cells. Human HIV-Nef protein, for example, is known to enhance the
activity
of a peroxisomal acyl-CoA thioesterase, PTEl, by a mechanism with involves the
binding of Nef to PTEl (Watanabe et al. (1997) Biochim. Biophys. Res Comm.
238:
234-239.
Preferably acyl-CoA thioesterase expression is modulated in a plant by
transforming the plant or plant cell with at least a portion of an acyl-CoA
thioesterase
nucleotide sequence. By "acyl-CoA thioesterase nucleotide sequence" is
intended any
nucleotide sequence of an acyl-CoA thioesterase gene or transcript, including,
but not
limited, to coding sequences, exons, introns, 5'-regulatory regions, 3'-
regulatory
regions, 5'-untranslated regions, 3'-untranslated regions and the like. Any
acyl-CoA
thioesterase nucleotide sequences known in the art may be employed in the
methods
of the present invention. Preferred acyl-CoA thioesterases nucleotide
sequences axe
those which encode peroxisomal acyl-CoA thioesterases. More preferred acyl-CoA
thioesterase nucleotide sequences include the nucleotide sequence set forth in
SEQ ID
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NO: 1, a nucleotide sequence comprising at least 45% identity to the
nucleotide set
forth in SEQ ID NO: l, a nucleotide sequence encoding the amino acid sequence
set
forth in SEQ ID NO: 2 and the nucleotide sequences set forth in GenBank
Accession
Nos. AF124264 and AF124265. Most preferred acyl-CoA thioesterases nucleotide
S sequences include the nucleotide sequence set forth in SEQ ID NO: 1, a
nucleotide
sequence comprising at least 45% identity to the nucleotide set forth in SEQ
ID NO: 1
and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID
NO:
2.
The methods of the invention can be used to increase the level of unusual
fatty
acyl chains in oil extracted from a seed that synthesizes such fatty acyl
chains during
seed development. In engineering crop plants to produce specialty oils
comprised of
triacylglcerols with one or more unusual fatty acyl chains, scientists have
encountered
difficulties in achieving desired levels of such molecules in the oil. While
it remains
unclear why such molecules are not produced at the desired level, the unusual
fatty
acyl molecules may be preferentially shunted toward J3-oxidation because such
molecules axe not generally preferred for esterification of glycerol skeletons
in
triacylglycerol biosynthesis. It is known that the first two acyltransferases
in
triacylglycerol biosynthesis have a rather narrow substrate specificity
(Harwood
(1996) Biochim Biophys Acta 1301:7-56), and thus, may act in vivo to limit the
incorporation of unusual fatty acyl chains into triacylglycerols. The present
invention
does not depend on a particular biological mechanism, only that decreasing or
increasing aryl-CoA thioesterase decreases /3-oxidation.
The methods for decreasing (3-oxidation increase the level of the desired
unusual fatty acyl-CoA for triacylglycerol biosynthesis. An increased level of
unusual fatty acyl chains in the developing seed increases the level of such
acyl chains
in triacylglycerol. In the presence of an increased level of unusual fatty
acids, the
enzyme diacylglycerol acyltransferase is known to take up these fatty acyl-
CoAs and
incorporate them into the sh-3 position of triacylglycerol. Thus, an increased
level of
an unusual fatty acyl chain can be achieved in seed oil.
In an embodiment of the invention, the level of unusual fatty acyl chains in
the
oil of a plant is increased. Any plant may be utilized that is capable of
producing
unusual fatty aryl chains in its seeds. Such a plant has been genetically
engineered to
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produce the desired fatty acyl chain in its seeds or may be a plant that
produces the
desired fatty acyl chain in the absence of recombinant DNA in its genome.
Preferably, such a plant is a crop plant, more preferably such a plant is crop
plant that
produces seeds that may be used for commercial oiI production such as, for
example,
soybean, corn, B~assica, sunflower, safflower, peanut, cotton, flax, castor
and palm.
To increase the level of unusual fatty aryl chains in the seed oil, the plant
can be
transformed with an acyl-CoA thioesterase nucleotide sequence operably linked
to a
promoter that drives expression in the plant, particularly in the seeds of the
plant.
Methods are provided for optimizing a plant for seed oil production. By
"optimizing a plant for seed oil production" is intended that a plant is
impacted in
such a manner as to favorably affect seed oil accumulation. That is, seed oil
accumulation is increased, at least one desired constituent of seed oil is
increased, or a
combination thereof. Thus, the methods find use in producing a plant that
accumulates in its seeds a desired level of oil, a desired level of an oil
constituent or
both. Such a plant is desired for the extraction of standard vegetable oils or
specialty
oils. The methods involve decreasing (3-oxidation by modulating acyl-CoA
thioesterase expression as described supra and additionally decreasing the
level or
activity of at least one additional protein involved in (3-oxidation in a
plant seed.
The invention does not depend on a particular additional protein, only that
decreasing or increasing the level or activity of such a protein in plant has
a desired
effect on seed oil accumulation in a plant. Generally, decreasing the level of
such a
protein will affect (3-oxidation in a plant or tissue thereof. A protein that
affects (3-
oxidation may be, for example, an enzyme in the ~3-oxidation pathway. Such
enzymes
include, but are not limited to, acyl-CoA oxidase, multifunctional protein
type II,
acyl-CoA synthetase, enoyl-CoA isomerase, ~i-ketoacyl-CoA thiolase and
hydroxyacyl-CoA epimerase. Other proteins include, but are not limited to,
catalase,
malate dehydrogenase, carnatine acetyl transferase, citrate synthase,
aconitase,
isocitrate lyase, malate synthetase, and acyl-CoA thioesterase. Preferred
proteins of
the invention include acyl-CoA oxidase and multifunctional protein type II.
The level or activity of the additional protein may be decreased by any method
known in the art. Such methods include, but are not limited to, cosuppression,
antisense suppression and chimeraplasty. Preferably, the level or activity of
the
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additional protein is decreased by transforming a plant with at least a
portion of
nucleotide construct that comprises a nucleotide sequence of a gene or
transcript
encoding the additional protein.
In producing high levels of seed oil, particularly seed oil comprised of
unusual
fatty acyl chains, further steps to decrease ~3-oxidation may be employed to
achieve
the desired level of oil. Because unusual fatty acyl-CoAs are not preferred as
substrates by the acyltransferases that transfer fatty acyl chains to the
glycerol moiety
in triacylglycerol synthesis, such fatty acyl chains are preferentially
shunted to [3-
oxidation in the developing seed. Decreasing ~3-oxidation in the developing
seed can
increase the pool of unusual fatty acyl chains available for triacylglycerol
synthesis.
It is known that in the presence of a high amount of unusual fatty acids, the
enzyme
diacylglycerol acyltransferase can incorporate such fatty acids into the sn-3
position
of triacylglycerol. Thus, the methods of the invention can provide seeds that
produce
oil with higher levels of triacylglycerols with unusual fatty acyl chains.
In preferred methods of the invention that involve decreasing the level of at
least one additional protein that affects j3-oxidation, a plant or plant cell
is transformed
with a nucleotide construct comprising a promoter operably linked to
nucleotide
sequence encoding such an additional protein. Preferably, the nucleotide
sequence
comprises a coding sequence for an acyl-CoA thioesterase or a multifunctional
protein type II. The methods of the invention additionally involve
cosuppression or
antisense suppression methods to decrease the level of at least one protein
involved in
~3-oxidation.
To determine that the desired increase in the content of oil and/or
triacylglycerols with the unusual fatty acyl chains has been achieved, oil may
be
extracted from seeds to determine total oil production and individual
constituents of
the oil may then be measured using any methods known to those of ordinary
skill in
the art.
The methods of the invention find use in increasing the level of unusual fatty
acyl acids or constituents of oil comprising acyl chains of such fatty acids.
By
"unusual" fatty acids is intended fatty acids that have structural features
such as, for
example, an epoxy group, a triple bond, methyl branching or unusual carbon-
chain
length. Such "unusual" fatty acids include, but are not limited to, vernolic
acid,
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petroselinic acid, sterculic acid, lesquerolic acid, densipolic acid,
auricolic acid, cis-5-
eicosenoic acid, cis-5-docosenoic acid, cis-5,13-docosdienoic acid,
chaulmoogric
acid, erucic acid, ricinoleic acid, labellenic acid, crepenynic acid and
stearolic acid.
The invention also encompasses increasing the level of one or more of the
acyltransferases that are involved in the biosynthesis of triacylglycerol such
as, for
example, the diacylglycerol acyltransferase from mouse (Accession No.
AF078752).
While the invention encompasses the use of any acyltransferase, preferred
acyltransferases are those that have a wide substrate range. Of particular
interest are
acyltransferases that can efficiently catalyze the esterification of at least
one unusual
fatty acid to the glycerol moiety.
The invention is drawn to methods which increase the level of oil or a
constituent thereof in a plant. Use of the term "oil" is not meant to limit
the invention
to fats or lipids that are liquid at room temperature, about 20°C to
25°C. The oil of
the present invention may or may not be liquid at room temperature. Generally,
if the
oil is solid at room temperature, heat may be applied to liquefy it.
The methods of the invention involve transforming a plant with a nucleotide
construct. Such methods involve the nucleotide construct gaining access to the
interior of a cell. The methods of the invention do not depend on a particular
method
for transforming a plant with a nucleotide construct, only that the nucleotide
construct
gains access to the interior of at least one cell of the plant. Methods for
transforming
plants are known in the art including, but not limited to, stable
transformation
methods and transient transformation methods
By "stable transformation" is intended that the nucleotide construct that is
introduced into a plant stably integrates into the genome of the plant is
capable of
being inherited by progeny thereof. By "transient transformation" is intended
that a
nucleotide construct that is introduced into a plant does not stably integrate
into the
genome of the plant.
In certain embodiments of the invention, a plant cell is transformed with at
least one nucleotide construct comprising a nucleotide sequence encoding an
acyl-
CoA thioesterase, or other protein affecting (3-oxidation, operably linked in
a sense or
antisense orientation to a promoter that drives expression in a plant cell.
Preferably,
such a transformed plant cell is regenerated into a stably transformed plant,
wherein
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the nucleotide construct is stably incorporated into the genome of the
transformed
plant.
The methods of the invention also encompass the use of the nucleotide
constructs of the invention in methods for altering or mutating any genomic
nucleotide sequence in an organism, such as, fox example, chimeraplasty. Thus,
the
methods of the invention may involve modulating acyl-CoA thioesterases genes
and
as well as other genes which might affect [3-oxidation in a plant. Airy part
of gene
may be altered including, but not limited to, nucleotide sequences of exons,
introns,
untranscribed regions of genes, regions corresponding to untranslated and
regions of a
transcript. For example, chimeraplasty may be used with an acyl-CoA nucleotide
sequence to increase or decrease acyl-CoA thioesterase expression in a plant
by
altering a coding sequence in the genome of the plant in such a manner that
the amino
acid sequence of the encoded acyl-CoA is changed.
Compositions of the invention include nucleotide sequences that encode acyl-
CoA thioesterases that are involved in (3-oxidation. In particular, the
present
invention provides for isolated nucleic acid molecules comprising nucleotide
sequences encoding the amino acid sequences shown in SEQ ID NO: 2. Further
provided are polypeptides having an amino acid sequence encoded by a nucleic
acid
molecule described herein, for example those set forth in SEQ ID NO: l, and
fragments and variants thereof.
The invention encompasses isolated or substantially puxified nucleic acid or
protein compositions. An "isolated" or "purified" nucleic acid molecule or
protein, or
biologically active portion thereof, is substantially free of other cellular
material, or
culture medium when produced by recombinant techniques, or subsfiantially free
of
chemical precursors or other chemicals when chemically synthesized.
Preferably, an
"isolated" nucleic acid is free of sequences (preferably protein encoding
sequences)
that naturally flank the nucleic acid (i.e., sequences located at the 5' and
3' ends of the
nucleic acid) in the genomic DNA of the organism from which the nucleic acid
is
derived. For example, in various embodiments, the isolated nucleic acid
molecule can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide
sequences that naturally flank the nucleic acid molecule in genomic DNA of the
cell
from which the nucleic acid is derived. A protein that is substantially free
of cellular
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material includes preparations of protein having less than about 30%, 20%,
10%, 5%,
(by dry weight) of contaminating protein. When the protein of the invention or
biologically active portion thereof is recombinantly produced, preferably
culture
medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of
chemical precursors or non-protein-of interest chemicals.
Fragments and variants of the disclosed nucleotide sequences and proteins
encoded thereby are also encompassed by the present invention. By "fragment"
is
intended a portion of the nucleotide sequence or a portion of the amino acid
sequence
and hence protein encoded thereby. Fragments of a nucleotide sequence may
encode
protein fragments that retain the biological of the native protein and hence
retain
acyl-CoA thioesterase activity. Alternatively, fragments of a nucleotide
sequence that
are useful as hybridization probes generally do not encode fragment proteins
retaining
biological activity. Thus, fragments of a nucleotide sequence may range from
at least
about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to
the full-
length nucleotide sequence encoding the proteins of the invention.
A fragment of an acyl-CoA thioesterase nucleotide sequence that encodes a
biologically active portion of an acyl-CoA thioesterase of the invention will
encode at
least 15, 25, 30, 50, 100, 150, 200, or 240 contiguous amino acids, or up to
the total
number of amino acids present in a full-length acyl-CoA thioesterase of the
invention
(for example, 242 amino acids for SEQ ID NO: 2). Fragments of an acyl-CoA
thioesterase nucleotide sequence that are useful as hybridization probes or
PCR
primers generally need not encode a biologically active portion of an acyl-CoA
thioesterase.
Thus, a fragment of an acyl-CoA thioesterase nucleotide sequence may encode
a biologically active portion of an acyl-CoA thioesterase, or it may be a
fragment that
can be used as a hybridization probe or PCR primer using methods disclosed
below.
A biologically active portion of an acyl-CoA thioesterase can be prepaxed by
isolating
a portion of one of the acyl-CoA thioesterase nucleotide sequences of the
invention,
expressing the encoded portion of the acyl-CoA thioesterase (e.g., by
recombinant
expression in vitro), and assessing the activity of the encoded portion of the
acyl-CoA
thioesterase. Nucleic acid molecules that are fragments of an acyl-CoA
thioesterase
nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300,
350,
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400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, or 1,100 nucleotides, or
up to the
number of nucleotides present in a full-length acyl-CoA thioesterase
nucleotide
sequence disclosed herein (for example, 1169 nucleotides for SEQ TD NO: 1)
By "variants" is intended substantially similar sequences. For nucleotide
sequences, conservative variants include those sequences that, because of the
degeneracy of the genetic code, encode the amino acid sequence of one of the
acyl-
CoA thioesterase polypeptides of the invention. Naturally occurring allelic
variants
such as these can be identified with the use of well-known molecular biology
techniques, as, for example, with polymerase chain reaction (PCR) and
hybridization
techniques as outlined below. Vaxiant nucleotide sequences also include
synthetically
derived nucleotide sequences, such as those generated, for example, by using
site-
directed mutagenesis but which still encode an the acyl-CoA thioesterase
protein of
the invention. Generally, variants of a particular nucleotide sequence of the
invention
will have at least about 40%, 50%, 60%, 65%, 70%, generally at least about
75%,
80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
and more preferably at least about 98%, 99% or more sequence identity to that
particular nucleotide sequence as determined by sequence aligmnent programs
described elsewhere herein using default parameters.
By "variant" protein is intended a protein derived from the native protein by
deletion (so-called truncation) or addition of one or more amino acids to the
N-
terminal and/or C-terminal end of the native protein; deletion or addition of
one or
more amino acids at one or more sites in the native protein; or substitution
of one or
more amino acids at one or more sites in the native protein. Variant proteins
encompassed by the present invention are biologically active, that is they
continue to
possess the desired biological activity of the native protein, that is, the
acyl-CoA
thioesterase activity as described herein. Such variants may result from, fox
example,
genetic polymorphism or from human manipulation. Biologically active variants
of a
native the acyl-CoA thioesterase protein of the invention will have at least
about 40%,
50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at
least
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least
about 98%, 99% or more sequence identity to the amino acid sequence for the
native
protein as determined by sequence alignment programs described elsewhere
herein
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using default parameters. A biologically active variant of a protein of the
invention
may differ from that protein by as few as 1-15 amino acid residues, as few as
1-10,
such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
The proteins of the invention may be altered in various ways including amino
acid substitutions, deletions, truncations, and insertions. Methods for such
manipulations axe generally known in the art. For example, amino acid sequence
variants of the acyl-CoA thioesterase can be prepared by mutations in the DNA.
Methods for mutagenesis and nucleotide sequence alterations are well known in
the
art. See, for example, Kunkel (1985) P~oc. Natl. Acad. Sci. USA 82:488-492;
Kunkel
et al. (1987) Methods in Enzymol. 154:367-382; US Patent No. 4,873,192; Walker
and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York) and the references cited therein. Guidance as to
appropriate
amino acid substitutions that do not affect biological activity of the protein
of interest
may be found in the model of Dayhoff et al. ( 1978) Atlas of P~otei~ Sequence
and
Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated
by
reference. ~ Conservative substitutions, such as exchanging one amino acid
with
another having similar properties, may be preferred.
Thus, the genes and nucleotide sequences of the invention include both the
naturally occurring sequences as well as mutant forms. Likewise, the proteins
of the
invention encompass both naturally occurring proteins as well as variations
and
modified forms thereof. Such variants will continue to possess the desired
acyl-CoA
thioesterase activity. Obviously, the mutations that will be made in the DNA
encoding the vaxiant must not place the sequence out of reading frame and
preferably
will not create complementary regions that could produce secondary mRNA
structure.
See, EP Patent Application Publication No. 75,444.
The deletions, insertions, and substitutions of the protein sequence
encompassed herein are not expected to produce radical changes in the
characteristics
of the protein. However, when it is difficult to predict the exact effect of
the
substitution, deletion, or insertion in advance of doing so, one skilled in
the art will
appreciate that the effect will be evaluated by routine screening assays. That
is, the
activity can be evaluated by assaying acyl-CoA thioesterase activity. See, for
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example, Jones et al. ((1999) J. Biol Chem. 274:9216-9223), herein
incorporated by
reference.
Variant nucleotide sequences and proteins also encompass sequences and
proteins
derived from a mutagenic and recombinogenic procedure such as DNA shuffling.
With such a procedure, one or more different acyl-CoA thioesterase coding
sequences
can be manipulated to create a new acyl-CoA thioesterase possessing the
desired
properties. In this manner, libraries of recombinant polynucleotides are
generated
from a population of related sequence polynucleotides comprising sequence
regions
that have substantial sequence identity and can be homologously recombined in
vitro
or in vivo. For example, using this approach, sequence motifs encoding a
domain of
interest may be shuffled between the acyl-CoA thioesterase gene of the
invention and
other known aryl-CoA thioesterase genes to obtain a new gene coding for a
protein
with an improved property of interest, such as an increased Km in the case of
an
enzyme. Strategies for such DNA shuffling are known in the art. See, for
example,
Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994)
Nature
370:389-391; Crarneri et al. (1997) Nature Biotech. 15:436-438; Moore et al.
(1997)
J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-
4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Patent Nos. 5,605,793
and
5,837,458.
The nucleotide sequences of the invention can be used to isolate
corresponding sequences from other organisms, particularly other plants, more
particularly other monocots. In this manner, methods such as PCR,
hybridization, and
the like can be used to identify such sequences based on their sequence
homology to
the sequence set forth herein. Sequences isolated based on their sequence
identity to
the entire acyl-CoA thioesterase sequence set forth herein or to fragments
thereof are
encompassed by the present invention. Such sequences include sequences that
are
orthologs of the disclosed sequences. By "orthologs" is intended genes derived
from
a common ancestral gene and which are found in different species as a result
of
speciation. Genes found in different species are considered orthologs when
their
nucleotide sequences and/or their encoded protein sequences share substantial
identity
as defined elsewhere herein. Functions of orthologs are often highly conserved
among species.
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In a PCR approach, oligonucleotide primers can be designed for use in PCR
reactions to amplify corresponding DNA sequences from cDNA or genomic DNA
extracted from any plant of interest. Methods for designing PCR primers and
PCR
cloning are generally known in the art and are disclosed in Sambrook et al.
(1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols:
A
Guide to Methods and Applications (Academic Press, New York); Innis and
Gelfand,
eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand,
eds.
(1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR
include, but are not limited to, methods using paired primers, nested primers,
single
specific primers, degenerate primers, gene-specific primers, vector-specific
primers,
partially-mismatched primers, and the like.
In hybridization techniques, all or part of a known nucleotide sequence is
used
as a probe that selectively hybridizes to other corresponding nucleotide
sequences
present in a population of cloned genomic DNA fragments or cDNA fragments
(i.e.,
genomic or cDNA libraries) from a chosen organism. The hybridization probes
may
be genomic DNA fragments, cDNA fragments, RNA fragments, or other
oligonucleotides, and may be labeled with a detectable group such as 32P, or
any other
detectable marker. Thus, for example, probes for hybridization can be made by
labeling synthetic oligonucleotides based on the acyl-CoA thioesterase
nucleotide
sequence of the invention. Methods for preparation of probes for hybridization
and for
construction of cDNA and genomic libraries are generally known in the art and
axe
disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, New York).
For example, the entire acyl-CoA thioesterase nucleotide sequence disclosed
herein, or one or more portions thereof, may be used as a probe capable of
specifically
hybridizing to corresponding acyl-CoA thioesterase nucleotide sequences [and
messenger RNAs]. To achieve specific hybridization under a variety of
conditions,
such probes include sequences that are unique among acyl-CoA thioesterase
nucleotide sequences and are preferably at least about 10 nucleotides in
length, and
most preferably at least about 20 nucleotides in length. Such probes may be
used to
amplify corresponding aryl-CoA thioesterase nucleotide sequences from a chosen
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plant by PCR. This technique may be used to isolate additional coding
sequences
from a desired plant or as a diagnostic assay to determine the presence of
coding
sequences in a plant. Hybridization techniques include hybridization screening
of
plated DNA libraries (either plaques or colonies; see, for example, Sambrook
et al.
(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, New York).
Hybridization of such sequences may be carried out under stringent
conditions. By "stringent conditions" or "stringent hybridization conditions"
is
intended conditions under which a probe will hybridize to its target sequence
to a
detectably greater degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will be different
in
different circumstances. By controlling the stringency of the hybridization
and/or
washing conditions, target sequences that are 100% complementary to the probe
can
be identified (homologous probing). Alternatively, stringency conditions can
be
adjusted to allow some mismatching in sequences so that lower degrees of
similarity
are detected (heterologous probing). Generally, a probe is less than about
1000
nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration
is
less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion
concentration (or
other salts) at pH 7.0 to 8.3 and the temperature is at least about
30°C for short probes
(e.g., 10 to 50 nucleotides) and at least about 60°C for long probes
(e.g., greater than
50 nucleotides). Stringent conditions may also be achieved with the addition
of
destabilizing agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI,
1%
SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1X to 2X SSC (20X
SSC = 3.0
M NaCI/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate
stringency
conditions include hybridization in 40 to 45% formamide, 1.0 M NaCI, 1% SDS at
37°C, and a wash in 0.5X to 1X SSC at 55 to 60°C. Exemplary high
stringency
conditions include hybridization in 50% formamide, 1 M NaCl, 1 % SDS at
37°C, and
a wash in O.IX SSC at 60 to 65°C. The duration of hybridization is
generally less
than about 24 hours, usually about 4 to about I2 hours
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Specificity is typically the function of post-hybridization washes, the
critical
factors being the ionic strength and temperature of the final wash solution.
For DNA-
DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl
(1984) Anal. Biochem.13~:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41
(%GC) -
0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is
the
percentage of guanosine and cytosine nucleotides in the DNA, % form is the
percentage of formamide in the hybridization solution, and L is the length of
the
hybrid in base pairs. The Tm is the temperature (under defined ionic strength
and pH)
atwhich 50% of a complementary target sequence hybridizes to a perfectly
matched
probe. Tm is reduced by about 1 °C for each 1 % of mismatching; thus,
Tm,
hybridization, and/or wash conditions can be adjusted to hybridize to
sequences of the
desired identity. For example, if sequences with >90% identity are sought, the
Tm can
be decreased 10°C. Generally, stringent conditions are selected to be
about 5°C lower
than the thermal melting point (Tm) for the specific sequence and its
complement at a
defined ionic strength and pH. However, severely stringent conditions can
utilize a
hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal
melting point (Tm);
moderately stringent conditions can utilize a hybridization and/or wash at 6,
7, 8, 9, or
10°C lower than the thermal melting point (Tm); low stringency
conditions can utilize
a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than
the thermal
melting point (Tm). Using the equation, hybridization and wash compositions,
and
desired Tm, those of ordinary skill will understand that variations in the
stringency of
hybridization and/or wash solutions are inherently described. If the desired
degree of
mismatching results in a Tm of less than 45°C (aqueous solution) or
32°C (formamide
solution), it is preferred to increase the SSC concentration so that a higher
temperature
can be used. An extensive guide to the hybridization of nucleic acids is found
in
Tijssen (1993) Laboratory Techniques ivc Biochemistry and Molecular Biology-
Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New
York); and
Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2
(Greene Publishing and Wiley-Interscience, New York). See Sambrook et al.
(1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, New York).
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Thus, isolated sequences that encode for an acyl-CoA thioesterase and which
hybridize under stringent conditions to the acyl-CoA thioesterase nucleotide
sequence
disclosed herein, or to fragments thereof, are encompassed by the present
invention.
The following terms are used to describe the sequence relationships between
two or more nucleic acids or polynucleotides: (a) "reference sequence", (b)
"comparison window", (c) "sequence identity", (d) "percentage of sequence
identity",
and (e) "substantial identity".
(a) As used herein, "reference sequence" is a defined sequence used as a
basis for sequence comparison. A reference sequence may be a subset or the
entirety
of a specified sequence; for example, as a segment of a full-length cDNA or
gene
sequence, or the complete cDNA or gene sequence.
(b) As used herein, "comparison window" makes reference to a contiguous
and specified segment of a polynucleotide sequence, wherein the polynucleotide
sequence in the comparison window may comprise additions or deletions (i.e.,
gaps)
compared to the reference sequence (which does not comprise additions or
deletions)
for optimal alignment of the two sequences. Generally, the comparison window
is at
least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50,
100, or
longer. Those of skill in the art understand that to avoid a high similarity
to a
reference sequence due to inclusion of gaps in the polynucleotide sequence a
gap
penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well known in the art.
Thus, the determination of percent identity between any two sequences can be
accomplished using a mathematical algoritlnn. Non-limiting examples of such
mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS
4:11-
17; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math.
2:482; the
homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol.
48:443-453; the search-for-similarity-method of Pearson and Lipman (1988)
P~oc.
Natl. Aced. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)
P~oc.
Natl. Aced. Sci. USA 872264, modified as in Karlin and Altschul (1993) P~oc.
Natl.
Acad. Sci. USA 90:5873-5877.
Computer implementations of these mathematical algorithms can be utilized
for comparison of sequences to determine sequence identity. Such
implementations
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include, but are not limited to: CLUSTAL in the PC/Gene program (available
from
Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0)
and
GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science
Drive, Madison, Wisconsin, USA). Alignments using these programs can be
performed using the default parameters. The CLUSTAL program is well described
by
Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS
5:151-
153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)
CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The
ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A
PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of
4 can
be used with the ALIGN program when comparing amino acid sequences. The
BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 axe based on the
algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can
be
performed with the BLASTN program, score = 100, wordlength = 12, to obtain
nucleotide sequences homologous to a nucleotide sequence encoding a protein of
the
invention. BLAST protein searches can be performed with the BLASTX program,
score = 50, wordlength = 3, to obtain amino acid sequences homologous to a
protein
or polypeptide of the invention. To obtain gapped alignments for comparison
purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul
et
al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0)
can be used to perform an iterated search that detects distant relationships
between
molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped
BLAST,
PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for
nucleotide sequences, BLASTX for proteins) can be used. See
http://www.ncbi.hlm.nih.gov. Alignment may also be performed manually by
inspection. Alignment may also be performed manually by inspection.
Unless otherwise stated, sequence identity/similarity values provided herein
refer to the value obtained using GAP Version 10 using the following
parameters:
identity using GAP Weight of 50 and Length Weight of 3; % similarity using Gap
Weight of 12 and Length Weight of 4, or any equivalent program. By "equivalent
program" is intended any sequence comparison program that, for any two
sequences
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in question, generates an aligmnent having identical nucleotide or amino acid
residue
matches and an identical percent sequence identity when compared to the
corresponding alignment generated by the preferred program.
GAP uses the algoritlun of Needleman and Wunsch (1970) J. Mol. Biol. 48:
443-453, to find the alignment of two complete sequences that maximizes the
number
of matches and minimizes the number of gaps. GAP considers all possible
alignments
and gap positions and creates the alignment with the largest number of matched
bases
and the fewest gaps. It allows for the provision of a gap creation penalty and
a gap
extension penalty in units of matched bases. GAP must make a profit of gap
creation
penalty number of matches for each gap it inserts. If a gap extension penalty
greater
than zero is chosen, GAP must, in addition, make a profit for each gap
inserted of the
length of the gap times the gap extension penalty. Default gap creation
penalty values
and gap extension penalty values in Version 10 of the Wisconsin Genetics
Software
Package for protein sequences are 8 and 2, respectively. For nucleotide
sequences
the default gap creation penalty is 50 while the default gap extension penalty
is 3.
The gap creation and gap extension penalties can be expressed as an integer
selected
from the group of integers consisting of from 0 to 200. Thus, for example, the
gap
creation and gap extension penalties can be 0, l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65 or greater.
GAP presents one member of the family of best alignments. There may be
many members of this family, but no other member has a better quality. GAP
displays four figures of merit for alignments: Quality, Ratio, Identity, and
Similarity.
The Quality is the metric maximized in order to align the sequences. Ratio is
the
quality divided by the number of bases in the shorter segment. Percent
Identity is the
percent of the symbols that actually match. Percent Similarity is the percent
of the
symbols that are similar. Symbols that are across from gaps are ignored. A
similarity
is scored when the scoring matrix value for a pair of symbols is greater than
or equal
to 0.50, the similarity threshold. The scoring matrix used in Version 10 of
the
Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff
(1989) Proc. Natl. Acad. Sci. USA 89:10915).
(c) As used herein, "sequence identity" or "identity" in the context of two
nucleic acid or polypeptide sequences makes reference to the residues in the
two
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sequences that are the same when aligned for maximum correspondence over a
specified comparison window. When percentage of sequence identity is used in
reference to proteins it is recognized that residue positions which are not
identical
often differ by conservative amino acid substitutions, where amino acid
residues are
substituted for other amino acid residues with similar chemical properties
(e.g., charge
or hydrophobicity) and therefore do not change the functional properties of
the
molecule. When sequences differ in conservative substitutions, the percent
sequence
identity may be adjusted upwards to correct for the conservative nature of the
substitution. Sequences that differ by such conservative substitutions are
said to have
"sequence similarity" or "similarity". Means for making this adjustment are
well
known to those of skill in the art. Typically this involves scoring a
conservative
substitution as a partial rather than a full mismatch, thereby increasing the
percentage
sequence identity. Thus, for example, where an identical amino acid is given a
score
of 1 and a non-conservative substitution is given a score of zero, a
conservative
substitution is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program PC/GENE
(Intelligenetics, Mountain View, California).
(d) As used herein, "percentage of sequence identity" means the value
determined by comparing two optimally aligned sequences over a comparison
window, wherein the portion of the polynucleotide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) as compared to the
reference
sequence (which does not comprise additions or deletions) for optimal
alignment of
the two sequences. The percentage is calculated by determining the number of
positions at which the identical nucleic acid base or amino acid residue
occurs in both
sequences to yield the number of matched positions, dividing the number of
matched
positions by the total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence identity.
(e)(i) The term "substantial identity" of polynucleotide sequences means that
a polynucleotide comprises a sequence that has at least 70% sequence identify,
preferably at least 80%, more preferably at least 90%, and most preferably at
least
95%, compaxed to a reference sequence using one of the alignment programs
described using standard parameters. One of skill in the art will recognize
that these
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values can be appropriately adjusted to determine corresponding identity of
proteins
encoded by two nucleotide sequences by taking into account codon degeneracy,
amino acid similarity, reading frame positioning, and the like. Substantial
identity of
amino acid sequences for these purposes normally means sequence identity of at
Ieast
60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.
Another indication that nucleotide sequences are substantially identical is if
two molecules hybridize to each other under stringent conditions. Generally,
stringent conditions are selected to be about 5°C lower than the
thermal melting point
(Tin) for the specific sequence at a defined ionic strength and pH. However,
stringent
conditions encompass temperatures in the range of about 1 °C to about
20°C lower
than the Tm, depending upon the desired degree of stringency as otherwise
qualified
herein. Nucleic acids that do not hybridize to each other under stringent
conditions
are still substantially identical if the polypeptides they encode are
substantially
identical. This may occur, e.g., when a copy of a nucleic acid is created
using the
maximum codon degeneracy permitted by the genetic code. One indication that
two
nucleic acid sequences are substantially identical is when the polypeptide
encoded by
the first nucleic acid is immunologically cross reactive with the polypeptide
encoded
by the second nucleic acid.
(e)(ii) The term "substantial identity" in the context of a peptide indicates
that
a peptide comprises a sequence with at least 70% sequence identity to a
reference
sequence, preferably 80%, more preferably 85%, most preferably at least 90% or
95%
sequence identity to the reference sequence over a specified comparison
window.
Preferably, optimal alignment is conducted using the homology alignment
algorithm
of Needleman and Wunsch (1970) J. Mol. Biol. 4:443-453. An indication that two
peptide sequences are substantially identical is that one peptide is
immunologically
reactive with antibodies raised against the second peptide. Thus, a peptide is
substantially identical to a second peptide, for example, where the two
peptides differ
only by a conservative substitution. Peptides that are "substantially similar"
share
sequences as noted above except that residue positions that are not identical
may
differ by conservative amino acid changes.
The use of the term "nucleotide constructs" herein is not intended to limit
the
present invention to nucleotide constructs comprising DNA. Those of ordinary
skill
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in the art will recognize that nucleotide constructs, particularly
polynucleotides and
oligonucleotides, comprised of ribonucleotides and combinations of
ribonucleotides
and deoxyribonucleotides may also be employed in the methods disclosed herein.
The nucleotide constructs, nucleotide molecules and nucleotide sequences of
the
invention additionally encompass all complementary forms of such constructs,
molecules and sequences. Further, the nucleotide constructs, nucleotide
molecules
and nucleotide sequences of the present invention encompass all nucleotide
constructs, molecules and sequences which can be employed in the methods of
the
present invention for transforming plants including, but not limited to, those
comprised of deoxyribonucleotides, ribonucleotides and combinations thereof.
Such
deoxyribonucleotides and ribonucleotides include both naturally occurring
molecules
and synthetic analogues. The nucleotide constructs, nucleotide molecules and
nucleotide sequences of the invention also encompass all forms of nucleotide
constructs including, but not limited to, single-stranded forms, double-
stranded forms,
I S hairpins, stem-and-loop structures and the like.
Furthermore, it is recognized that the methods of the invention may employ a
nucleotide construct that is capable of directing, in a transformed plant, the
expression
of at least one protein, or at least one RNA, such as, for example, an
antisense RNA
that is complementary to at least a portion of an mRNA. Typically such a
nucleotide
construct is comprised of a coding sequence for a protein or an RNA operably
linked
to 5' and 3' transcriptional regulatory regions. Alternatively, it is also
recognized that
the methods of the invention may employ a nucleotide construct that is not
capable of
directing, in a transformed plant, the expression of a protein or an RNA.
In addition, it is recognized that methods of the present invention do not
depend on the incorporation of the entire nucleotide construct into the
genome, only
that the plant or cell thereof is altered as a result of the introduction of
the nucleotide
construct into a cell. In various embodiments of the invention, the genome of
a plant
is altered following the introduction of the nucleotide construct into a cell.
For
example, the nucleotide construct, or any part thereof, may incorporate into
the
genome of the plant. Alterations to the genome of the present invention
include, but
are not limited to, additions, deletions and substitution of nucleotides in
the genome.
While the methods of the present invention do not depend on additions,
deletions, or
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substitutions of any particular number of nucleotides, it is recognized that
such
additions, deletions or substitutions comprise at least one nucleotide.
The nucleotide constructs of the invention also encompass nucleotide
constructs, that may be employed in methods for altering or mutating a genomic
nucleotide sequence in an organism, including, but not limited to, chimeric
vectors,
chimeric mutational vectors, chimeric repair vectors, mixed-duplex
oligonucleotides,
self complementary chimeric oligonucleotides and recombinogenic
oligonucleobases.
Such nucleotide constructs and methods of use, such as, for example,
chimeraplasty,
are known in the art. Chimeraplasty involves the use of such nucleotide
constructs to
introduce site-specific changes into the sequence of genomic DNA within an
organism. See, U.S. Patent Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012;
5,795,972; and 5,871,984; all of which are herein incorporated by reference.
See also,
WO 98/49350, WO 99/07865, WO 99/25821 and Beetham et al. (1999) Proc. Natl.
Acad. Sci. USA 96:8774-8778; herein incorporated by reference.
If necessary, an acyl-CoA thioesterase or other protein may be modified to
direct the protein to the peroxisomes of a plant. Methods for directing a
protein to the
peroxisome are well known in the art. Typically, such methods involve operably
linking a nucleotide sequence encoding a peroxisome-targeting signal to the
coding
sequence of the protein or modifying the coding sequence of the protein to
additionally encode the peroxisome-targeting signal without substantially
affecting
the intended function of the encoded protein. See, for example, Olsen et al.
(1993)
Plant Cell 5:941-952, Mullen et al. ( 1997) Plant Physiol. 115:881-889, Gould
et al.
(1990) EMBO J. 9:85-90, Flynn et al. (1998) Plant J. 16:709-720; Preisig-
Muller and
Kindl (1993) Plant Mol. Biol. 22:59-66 and Kato et al. (1996) Plat Cell 8:1601-
161 l; herein incorporated by reference.
It is recognized that an protein of the invention may be directed to the
peroxisorne by operably linking a peroxisome-targeting signal to the C-
terminus or
the N-terminus of the protein. It is further recognized that an protein which
is
synthesized with a peroxisome-targeting signal may be processed
proteolytically in
vivo resulting in the removal of the peroxisome-targeting signal from the
amino acid
sequence of the mature, peroxisome-localized protein.
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The acyl-CoA thioesterase nucleotide sequences of the invention axe provided
in expression cassettes for expression in the plant of interest. The cassette
will
include 5' and 3' regulatory sequences operably linked to an acyl-CoA
thioesterase
nucleotide sequence of the invention. By "operably linked" is intended a
functional
linkage between a promoter and a second sequence, wherein the promoter
sequence
initiates and mediates transcription of the DNA sequence corresponding to the
second
sequence. Generally, operably linked means that the nucleic acid sequences
being
linked are contiguous and, where necessary to join two protein coding regions,
contiguous and in the same reading frame. In the case of protein coding
sequences,
"operably linked" includes joining two protein coding sequences in such a
manner
that both sequences are in the same reading frame for translation. For
example, a
nucleotide sequence encoding a peroxisome-targeting signal may be joined to
the 3'
end of a coding sequence of a protein of the invention in such manner that
both
sequences are in the same reading frame for translation to yield a the protein
of the
invention with a C-terminal addition of the peroxisome-targeting signal.
The expression cassette may additionally contain at least one additional gene
to be cotransformed into the organism. Alternatively, the additional genes)
can be
provided on multiple expression cassettes.
Such an expression cassette is provided with a plurality of restriction sites
for
insertion of the acyl-CoA thioesterase nucleotide sequence to be under the
transcriptional regulation of the regulatory regions. The expression cassette
may
additionally contain selectable marker genes.
The expression cassette will include in the 5'-3' direction of transcription,
a
transcriptional and translational initiation region, an acyl-CoA thioesterase
DNA
sequence of the invention, and a transcriptional and translational termination
region
functional in plants. The transcriptional initiation region, the promoter, may
be native
or analogous or foreign or heterologous to the plant host. Additionally, the
promoter
may be the natural sequence or alternatively a synthetic sequence. By
"foreign" is
intended that the transcriptional initiation region is not found in the native
plant into
which the transcriptional initiation region is introduced. As used herein, a
chimeric
gene comprises a coding sequence operably linked to a transcription initiation
region
that is heterologous to the coding sequence. -
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While it may be preferable to express the sequences using heterologous
promoters, the native promoter sequences may be used. Such constructs would
change expression levels of acyl-CoA thioesterase in the plant or plant cell.
Thus, the
phenotype of the plant or plant cell is altered.
The termination region may be native with the transcriptional initiation
region,
may be native with the operably linked DNA sequence of interest, or may be
derived
from another source. Convenient termination regions are available from the Ti-
plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase
termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-
144;
Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;
Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-
158;
Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987)
Nucleic
Acid Res. 15:9627-9639.
Where appropriate, the genes) may be optimized for increased expression in
the transformed plant. That is, the genes can be synthesized using plant-
preferred
codons for improved expression. See, for example, Campbell and Gowri (1990)
Plant
Physiol. 92:I-11 for a discussion of host-preferred codon usage. Methods are
available in the art for synthesizing plant-preferred genes. See, for example,
U.S.
Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids
Res.
17:477-498, herein incorporated by reference.
Additional sequence modifications are known to enhance gene expression in a
cellular host. These include elimination of sequences encoding spurious
polyadenylation signals, exon-intron splice site signals, transposon-like
repeats, and
other such well-characterized sequences that may be deleterious to gene
expression.
The G-C content of the sequence may be adjusted to levels average for a given
cellular host, as calculated by reference to known genes expressed in the host
cell.
When possible, the sequence is modified to avoid predicted hairpin secondary
mRNA
structures.
The expression cassettes may additionally contain 5' leader sequences in the
expression cassette construct. Such leader sequences can act to enhance
translation.
Translation leaders are known in the art and include: picornavirus leaders,
for
example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein
et
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CA 02414408 2003-O1-03
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al. (1989) P~oc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for
example,
TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gehe 165(2):233-238),
MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human
immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature
353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic
virus
(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus
leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech
(Liss, New
York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et
al.
(1991) hi~ology 81:382-385). See also, Della-Cioppa et al. (1987) Plaht
Physiol.
84:965-968. Other methods known to enhance translation can also be utilized,
for
example, introns, and the like.
In preparing the expression cassette, the various DNA fragments may be
manipulated, so as to provide for the DNA sequences in the proper orientation
and, as
appropriate, in the proper reading frame. Toward this end, adapters or linkers
may be
employed to join the DNA fragments or other manipulations may be involved to
provide for convenient restriction sites, removal of superfluous DNA, removal
of
restriction sites, or the like. For this purpose, in vitro mutagenesis, primer
repair,
restriction, annealing, resubstitutions, e.g., transitions and transversions,
may be
involved.
A number of promoters can be used in the practice of the invention. The
promoters can be selected based on the desired outcome. The nucleic acids can
be
combined with constitutive, tissue-preferred, or other promoters for
expression in
plants.
Such constitutive promoters include, fox example, the core promoter of the
Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and
U.S.
Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature
313:810-812); rice actin (McElroy et al. (1990) Plaht Cell x:163-171);
ubiquitin
(Christensen et al. (1989) Plat Mol. Biol. 12:619-632 and Christensen et al.
(1992)
PlantMol. Biol. 18:675-689); pEMU (Last et al. (1991) Theoi°. Appl.
Genet. 81:581-
588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S.
Patent
No. 5,659,026), and the like. Other constitutive promoters include, for
example, U.S.
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WO 02/04648 PCT/USO1/21458
PatentNos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;
5,268,463; and 5,608,142.
Chemical-regulated promoters can be used to modulate the expression of a
gene in a plant through the application of an exogenous chemical regulator.
Depending upon the objective, the promoter may be a chemical-inducible
promoter,
where application of the chemical induces gene expression, or a chemical-
repressible
promoter, where application of the chemical represses gene expression.
Chemical-
inducible promoters are known in the art and include, but are not limited to,
the maize
In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners,
the
maize GST promoter, which is activated by hydrophobic electrophilic compounds
that
are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is
activated by salicylic acid. Other chemical-regulated promoters of interest
include
steroid-responsive promoters (see, for example, the glucocorticoid-inducible
promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis
et
al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-
repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet.
227:229-
237, and LT.S. Patent Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
Tissue-preferred promoters can be utilized to target enhanced acyl-CoA
thioesterase expression within a particular plant tissue. Tissue-preferred
promoters
include Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al. (1997)
Plant
Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-
343;
Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996)
Plant
Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-
535;
Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al.
(1994)
Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.
20:181-
196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al.
(1993)
Proc Natl. Acad. Sci. USA 9000):9586-9590; and Guevara-Garcia et al. (1993)
Plant
J. 4(3):495-505. Such promoters can be modified, if necessary, for weak
expression.
Leaf preferred promoters are known in the art. See, for example, Yamamoto et
al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-
67;
Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993)
Plant
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WO 02/04648 PCT/USO1/21458
J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and
Matsuoka et
al. (1993) P~oc. Natl. Acad. Sci. USA 90(20):9586-9590.
Root-preferred promoters are known and can be selected from the many
available from the literature or isolated de novo from various compatible
species.
See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean
root-
specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-specif c control element in the GRP 1.8 gene of French
bean);
Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of
the
mannopine synthase (MAS) gene of Agrobacte~ium tumefaciens); and Miao et al.
(1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic
glutamine
synthetase (GS), which is expressed in roots and root nodules of soybean). See
also
Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-specific
promoters
isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia
andersonii and the related non-nitrogen-fixing nonlegume Trema tornentosa are
described. The promoters of these genes were linked to a (3-glucuronidase
reporter
gene and introduced into both the nonlegume Nicotiana tabacum and the legume
Lotus corniculatus, and in both instances root-specific promoter activity was
preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of
the
highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes
(see
Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-
preferred DNA determinants are dissociated in those promoters. Teeri et al.
(1989)
used gene fusion to lacZ to show that the Agrobacte~ium T-DNA gene encoding
octopine synthase is especially active in the epidermis of the root tip and
that the TR2'
gene is root specific in the intact plant and stimulated by wounding in leaf
tissue, an
especially desirable combination of characteristics for use with an
insecticidal or
larvicidal gene (see EMBO J. 8(2):343-350). The TRl' gene, fused to nptll
(neomycin phosphotransferase II) showed similar characteristics. Additional
root-
preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al.
(1995)
Plant Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994) Plant
Mol.
Biol. 25(4):681-691. See also U.S. Patent Nos. 5,837,876; 5,750,386;
5,633,363;
5,459,252; 5,401,836; 5,110,732; and 5,023,179.
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"Seed-preferred" promoters include both "seed-specific" promoters (those
promoters active during seed development such as promoters of seed storage
proteins)
as well as "seed-germinating" promoters (those promoters active during seed
germination). See Thompson et al. (1989) BioEssays 10:108, herein incorporated
by
reference. Such seed-preferred promoters include, but are not limited to, Ciml
(cytokinin-induced message); cZ19B1 (maize 19 kDa zero); milps (myo-inositol-1-
phosphate synthase); and celA (cellulose synthase) (see the copending
application
entitled "Seed-Preferred Promoters," U.S. Application Serial No. 60/097,233,
filed
August 20, 1998, herein incorporated by reference). Gama-zero is a preferred
endosperm-preferred promoter. Glob-1 is a preferred embryo-preferred promoter.
For dicots, seed-preferred promoters include, but are not limited to, bean (3-
phaseolin,
napin, (3-conglycinin, soybean lectin, cruciferin, and the like. For monocots,
seed-
preferred promoters include, but are not limited to, maize 15 kDa zero, 22 kDa
zero,
27 kDa zero, y-zero, waxy, shrunken 1, shrunken 2, globulin 1, etc.
Generally, the expression cassette will comprise a selectable marker gene for
the
selection of transformed cells. Selectable marker genes are utilized for the
selection of
transformed cells or tissues. Marker genes include genes encoding antibiotic
resistance,
such as those encoding neomycin phosphotransferase II (NEO) and hygromycin
phosphotransferase (HPT), as well as genes conferring resistance to herbicidal
compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-
dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Cur. Opin.
Biotech.
3:506-51 I; Christopherson et al. (I992) Proc. Natl. Acad. Sci. USA 89:6314-
6318; Yao
et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422;
Barkley et
al. (1980) in The Ope~on, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown
et al.
(1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al.
(1989)
Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl.
Acad. Sci.
USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)
Ph.D.
Thesis, University of Heidelberg; Refines et al. (1993) Proc. Natl. Acad. Sci.
USA
90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et
al.~
(1992) P~oc. Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl.
Acad.
Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al.
(1991)
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WO 02/04648 PCT/USO1/21458
Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988)
Biochemistry
27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et
al.
(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)
Antimicrob. Agents
Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental
Pharmacology,
Vol. 78 ( Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724.
Such
disclosures are herein incorporated by reference.
The above list of selectable marker genes is not meant to be limiting. Any
selectable marker gene can be used in the present invention.
Transformation protocols as well as protocols for introducing nucleotide
sequences into plants may vary depending on the type of plant or plant cell,
i.e.,
monocot or dicot, targeted for transformation. Suitable methods of introducing
nucleotide sequences into plant cells and subsequent insertion into the plant
genome
include microinjection (Crossway et al. (1986) Biotechniques 4:320-334),
electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,
Agrobacterium-mediated transformation (Townsend et al., U.S. Patent No.
5,563,055;
Zhao et al., U.S. Patent No. 5,981,840), direct gene transfer (Paszkowski et
al. (1984)
EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example,
Sanford
et al., U.S. Patent No. 4,945,050; Tomes et al., U.S. Patent No. 5,879,918;
Tomes et
al., U.S. Patent No. 5,886,244; Bidney et al., U.S. Patent No. 5,932,782;
Tomes et al.
(1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile
Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,
ed.
Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)
Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet.
22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37
(onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et
al.
(1988) BiolTechnology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro
Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.
96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice);
Klein et al.
(1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)
Biotechnology 6:559-563 (maize); Tomes, U.S. Patent No. 5,240,855; Buising et
al.,
U.S. Patent Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) 'Direct DNA
Transfer
into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell,
Tissue, and
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Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)
(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al.
(1990)
Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature
(London) 311:763-764; Bowen et al., U.S. Patent No. 5,736,369 (cereals);
Bytebier et
al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.
(1985)
in The Expef-imental Manipulation of Ovule Tissues, ed. Chapman et al.
(Longman,
New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports
9:415-
418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-
mediated
transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505
(electroporation); Li
et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995)
Annals of
Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750
(maize via Ag~obacterium tumefaciens); all of which are herein incorporated by
reference.
The cells that have been transformed may be grown into plants in accordance
with conventional ways. See, for example, McCormick et al. (1986) Plant Cell
Reports 5:81-84. These plants may then be grown, and either pollinated with
the
same transformed strain or different strains, and the resulting hybrid having
constitutive expression of the desired phenotypic characteristic identif ed.
Two or
more generations may be grown to ensure that expression of the desired
phenotypic
characteristic is stably maintained and inherited and then seeds harvested to
ensure
expression of the desired phenotypic characteristic has been achieved.
A plant can also be transformed with an acyl-CoA thioesterase nucleotide
construct or other nucleotide construct of the invention by contacting the
plant with a
virus or viral nucleic acids. Generally, such methods involve incorporating
the
nucleotide construct of interest within a viral DNA or RNA molecule. It is
recognized that the an acyl-CoA thioesterase of the invention may be initially
synthesized as part of a viral polyprotein which later may be processed by
proteolysis
in vivo or in vitro to produce the desired recombinant protein. Further, it is
recognized that promoters of the invention also encompass promoters utilized
for
transcription by viral RNA polymerases. Methods for introducing nucleotide
constn.2cts into plants and expressing a protein encoded therein involving
viral DNA
or RNA molecules are known in the art. See, for example, U.S. Patent Nos.
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5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated
by
reference.
It is recognized that with these nucleotide sequences, antisense
constructions,
complementary to at least a portion of the messenger RNA (mRNA) for the acyl-
CoA
thioesterase sequences can be constructed. Antisense nucleotides are
constructed to
hybridize with the corresponding mRNA. Modifications of the antisense
sequences
may be made as long as the sequences hybridize to and interfere with
expression of
the corresponding mRNA. In this manner, antisense constructions having 70%,
preferably 80%, more preferably 85% sequence identity to the corresponding
antisensed sequences may be used. Furthermore, portions of the antisense
nucleotides
may be used to disrupt the expression of the target gene. Generally, sequences
of at
least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be
used.
The nucleotide sequences of the present invention may also be used in the
sense orientation to suppress the expression of endogenous genes in plants.
Methods
1 S for suppressing gene expression in plants using nucleotide sequences in
the sense
orientation, often referred to as cosuppression methods, are known in the art.
Such
methods may be referred to methods The methods generally involve transforming
plants with a nucleotide construct comprising a promoter that drives
expression in a
plant operably linked to at least a portion of a nucleotide sequence that
corresponds to
the transcript of the endogenous gene. Typically, such a nucleotide sequence
has
substantial sequence identity to the sequence of the transcript of the
endogenous gene,
preferably greater than about 65% sequence identity, more preferably greater
than
about 85% sequence identity, most preferably greater than about 95% sequence
identity. See, Il.S. Patent Nos. 5,283,184 and 5,034,323; herein incorporated
by
' reference.
In the practice of certain specific embodiments of the present invention, a
plant or cell thereof may be transformed with two or more nucleotide
constructs.
Those of ordinary skill in the art realize that this can be accomplished in
any one of a
number of ways. For example, each of the respective nucleotide constructs may
be
introduced into a cell, or the constructs may be ligated together to form a
single
construct and then used to transform a cell. Alternatively, separate cell can
be
transformed with each of the separate constructs containing one or a subset of
the
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desired nucleotide constructs. Transformed plants that posses the desired
transgenic
phenotype can be regenerated and then selected by standard methods available
in the
art such as, for example, assaying enzyme activities, immunoblotting using
antibodies
which bind to the enzymes of interest, assaying for the products of a reporter
or
marker gene, and the like. Then, all of the desired nucleotide constructs can
be
brought together into a single plant through one or more rounds of cross
pollination
utilizing the previously selected transformed plants as parents.
Methods for cross pollinating plants are well known to those skilled in the
art,
and are generally accomplished by allowing the pollen of one plant, the pollen
donor,
to pollinate a flower of a second plant, the pollen recipient, and then
allowing the
fertilized eggs in the pollinated flower to mature into seeds. Progeny
containing the
entire complement of desired nucleotide constructs of the parental plants can
be
selected from the progeny by standard methods available in the art as
described supra
for selecting transformed plants. If necessary, the selected progeny can be
used as
either the pollen donor or pollen recipient in a subsequent cross pollination.
The present invention may be used for transformation of any plant species,
including, but not limited to, corn (Zea mays), B~assica spp. (e.g., B. napus,
B. rapa, B.
juncea), particularly those Brassica species useful as sources of seed oil,
alfalfa
(Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum
bicolor,
Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso
millet
(Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine
coracana)),
sunflower (Helianthus annuus), safflower (Ca~thamus tinctorius), wheat
(Ti~iticum
aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato
(Solarium
tuberosum), peanuts (Af~achis hypogaea), cotton (Gossypium barbadense,
Gossypium
hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee
(Coffea
spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees
(Citrus spp.),
cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado
(Persea americana), fig (Ficus casica), guava (Psidium guajava), mango
(Mangifera
indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anaca~dium
occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus),
sugar
beets (Beta vulga~is), sugarcane (Saccharum spp.), oats, barley, vegetables,
ornamentals,
and conifers.
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Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca
sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis),
peas
(Lathyrus spp.), and members of the genus Cucumis such as cucumber (C
sativus),
cantaloupe (C. cantalupensis), and musk melon (C melo). Ornamentals include
azalea
(Rhododendron spp.), hydrangea (MacroplZylla hydrangea), hibiscus (Hibiscus
rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus
spp.), petunias
(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrirna), and chrysanthemum. Conifers that may be employed in practicing
the
present invention include, for example, pines such as loblolly pine (Pinus
taeda), slash
pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus
contorta),
and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);
Western
hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia
sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir
(Abies
balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska
yellow-cedar (Chamaecyparis nootkatensis).
Plants of particular interest include grain plants that provide seeds of
interest,
oilseed plants, and leguminous plants. Seeds of interest include grain seeds,
such as
maize (corn), wheat, barley, rice, sorghum, rye, etc. Oilseed plants include
cotton,
soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
Leguminous plants include beans and peas. Beans include guar, locust bean,
fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean,
lentils,
chickpea, etc. Preferably, plants of the present invention are crop plants
(for example,
corn, soybean, rice, sunflower, Brassica, safflower, peanut, sorghum, wheat,
cotton,
millet, tobacco, alfalfa, etc.), more preferably corn and soybean plants, yet
more
preferably corn plants.
The allosteric effectors and enzyme inhibitors of the invention can be
administered to a plant in an effective amount by, for example, injecting,
infusing,
pipeting, spraying, atomizing, dusting, scattering, coating or pouring,
introducing into
or on the soil, introducing into irrigation water, by seed treatment or
general
application or dusting at any time. By "effective amount" is intended an
amount that,
when administered to a plant, produces the desired decrease or increase in
acyl-CoA
thioesterase activity.
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Oil or oil constituents can be measured by any method known in the art. In
order
to make a determination of the amount of oil in seeds and/or the amount of
specific fatty
acids present in the oil and their respective concentrations, mature seeds can
be crushed
(e.g., in a hydraulic press), and the endogenous oil can be readily extracted
with hexane
or by other suitable techniques in accordance with procedures known in the
art.
Similarly, any plant tissue can be ground or crushed and then extracted with
hexane to
recover oil. The hexane can be separated from the oil by evaporation, and the
amount of
oil remaining determined. The fatty acids can be determined following
transmethylation. The resulting methyl esters of the fatty acids can be
separated, and
their concentrations determined by use of capillary gas chromatography in
accordance
with standard operating procedures known in the art. For example, a Hewlett-
Packard
5890 gas chromatograph and a 7673 autosampler with a flame ionization detector
can be
utilized. The data can be collected and integrated using Perkin Elmer software
in
conjunction with Perkin Eliner interfaces. The integrated areas of the peaks
corresponding to the methyl esters of the various fatty acids can be grouped
and
normalized to yield their relative abundances. See "Automating Fatty Acid
Analyses
From Seeds" by, Thomas B. Brumback, Jr. et al., Chemiometrics and Intelligent
Laboratory Systems: Laboratory Information Management, Vol. 21, Page 215 to
222
(1993). Other analytical techniques similarly can be utilized that are known
to yield
reliable results, such as the American Oil Chemists Society (AOCS) Official
Method Ce
1e-91.
The following examples are presented by way of illustration, not by way of
limitation.
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EXPERIMENTAL
EXAMPLE 1
Manipulating oil biosynthesis and oil accumulation in plants
Acyl-CoA thioesterases catalyze the hydrolysis of acyl-CoA to free fatty acids
and CoA. They are found in the cytosol, mitochondria, and peroxisomes in yeast
and
animal cells (Smith (1994) FASEB J 8:1248-1259). The peroxisomal acyl-CoA
thioesterase (PTE) has been implicated in fatty acid (3-oxidation in yeast
because loss
of this gene interferes with the ability of yeast to grow on fatty acids
(Jones et al.
(1999) J. Biol. Chem. 274:9216-9223). Moreover, overexpression of this gene in
yeast also leads to reduced (3-oxidation (Chang et al. (1999) J. Cell Sci.
112:1579-
1590). Cosuppression and/or overexpression of this gene in plant cells would
likely
change the (3-oxidation rate and thereby reduce the flux of fatty acids
through
peroxisome.
A cDNA (SEQ ID NO: 1) of maize homolog to the yeast PTE gene has been
identified. A nucleotide construct comprising a nucleotide sequence of the
invention
operably linked to the oleosin promoter has been introduced into maize plants
for the
following objectives. First, this gene is useful in the production of unusual
fatty acids
such as vernolate in plants. Vernolate-producing Arabidopsis lines have been
reported to have a greater flux of fatty acids through the peroxisomes, which
may
account for the relatively low level (<3% seed weight) accumulation of this
fatty acid
in mature seeds. By reducing the peroxisomal (3-oxidation rate during seed
development, higher levels of the novel fatty acid can be accumulated. Second,
a
lower rate of (3-oxidation in seed crops during seed maturation can increase
the overall
oil accumulation.
Unusual fatty acids such as epoxy (vernolate) and hydroxy (ricinoleic) fatty
acids have a wide range of industrial applications (Gunstone et al. (1994) The
Lipid
Handbook, Chapman & Hall, London). Previous transgenic approaches to producing
large quantities of these fatty acids in high-yield grain crops encountered
low-level
accumulation of the desired fatty acid, and thus were not economically viable.
The
limitation placed on non-natural fatty acid accumulation in these crops may
stem from
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the fact that the acyltransferases for triacylglycerol (TAG) synthesis prefer
the natural
fatty acyl-CoAs to the unusual fatty acyl-CoA. It is well established that at
least the
first two acyltransferases in TAG biosynthesis have rather narrow substrate
specificity
(Harwood (1996) Biochim Biophys Acta 1301:7-56). Inefficient incorporation of
the
unusual fatty acid would then lead to increased flux of these fatty acids
through the
peroxisome to recycle the carbon for the synthesis of natural fatty acids. One
way to
overcome this is to reduce the (3-oxidation rate in plant cells and thereby
increase the
proportion of the desired fatty acyl-CoA in the acyl-CoA pool for TAG
synthesis. In
the presence of a high amount of unusual fatty acids, the acyltransferases
would be
forced to utilize such fatty acids, resulting in a higher levels of
accumulation of such
fatty acids in mature seeds.
The peroxisomal acyl-CoA thioesterase is believed to regulate the CoA pool in
peroxisomes. Fatty acids must be esterified with CoA before their (3-
oxidation.
However, if CoA is appended to poorly metabolized or nonmetabolizable fatty
acids,
CoASH levels would fall as more and more CoA is incorporated into these
metabolic
sinks. Peroxisomal acyl-CoA thioesterase serves to liberate the CoASH from
these
dead-end molecules (Chang et al. (1999) J. Cell Sci. 112:1579-1590). The
liberated
CoASH will be used to activate other fatty acids, while the released fatty
acid will be
free to equilibrate with cellular and extracellular pools, reducing their
concentration in
the peroxisome. Experimental evidence has shown that loss of function or over-
expression of a PTE gene leads to reduction in (3-oxidation and peroxisome
abundance. A PTE nucleotide sequence of the invention operably linked to a
seed-
preferred promoter can be used to transform Ar~abidopsis plants that produce
vernolate
in their seeds. Expressing this PTE gene in a seed-specific manner in
vernolate-
producing Arabidopsis plants can lead to increased PTE which can inhibit fatty
acid
oxidation by eliminating acyl-CoA substrates. The increased concentration of
the
desired fatty acid in the endoplasmic reticulum (ER) would shift the
equilibrium to
making TAG with enhanced contents of unusual fatty acids.
When expressed in maize, PTE can serve the same function to break up the
acyl-CoA substrates of (3-oxidation. The normal flux of fatty acids through
peroxisomes would then be re-equilibrated into other cellulax pools. At least
a portion
of these fatty acids are expected to be directed to the synthesis of TAG in
the ER. A
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number of other genes can also be used in combination with PTE. They include
the
acyl-CoA oxidase and the multifunctional protein type II. These proteins exert
metabolic control on [3-oxidation and peroxisome abundance (Chang et al.
(1999) J.
Cell Sci. 112:1579-1590). Cosuppression of the genes encoding one or both of
these
proteins can inhibit fatty acid [3-oxidation and increase the fatty acid flux
toward the
biosynthesis of TAG resulting in maize kernels with a increased content of
oil.
EXAMPLE 2
Transformation and Regeneration of Transgenic Maize Plants by Particle
Bombardment
Immature maize embryos from greenhouse donor plants are bombarded with a
plasmid containing the acyl-CoA thioesterase nucleotide sequence of the
invention
operably linked to a oleosin promoter plus a plasmid containing the selectable
marker
gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to
the
herbicide Bialaphos. Transformation is performed as follows. Media recipes
follow
below.
Preparation of Target Tissue
Immature embryos from Fl hybrid plants of the Hill genotype (Armstrong et
al. (1991) Maize Genet. Coop. Newslett. 65:92-93) are isolated 8-10 days after
pollination at 1.2-I.8 mm in length. The ears are husked and surface
sterilized in 30%
Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times
with
sterile water. The immature embryos are excised and placed embryo axis side
down
(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then
aligned within the 2.5-cm target zone in preparation for bombardment.
Preparation of DNA
A plasmid vector comprising the acyl-CoA thioesterase nucleotide sequence
of the invention operably linked to a oleosin promoter is made. This plasmid
DNA
plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1
~m
(average diameter) tungsten pellets using a CaCl2 precipitation procedure as
follows:
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100 ~,1 prepared tungsten particles in water
~l (1 ~,g) DNA in Tris EDTA buffer (1 pg total DNA)
100 ~,l 2.5 M CaC 12
5 10 ~,l 0.1 M spermidine
Each reagent is added sequentially to the tungsten particle suspension, while
maintained on the multitube vortexer. The final mixture is sonicated briefly
and
allowed to incubate under constant vortexing for 10 minutes. After the
precipitation
10 period, the tubes are centrifuged briefly, liquid removed, washed with 500
ml 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105
p,1
100% ethanol is added to the final tungsten particle pellet. For particle gun
bombardment, the tungstenlDNA particles are briefly sonicated and 10 ~,1
spotted
onto the center of each macrocarrier and allowed to dry about 2 minutes before
bombardment.
Bombardment and Culture Media
Bombardment medium (560Y) comprises 4.0 g/1 N6 basal salts (SIGMA C-
1416), 1.0 m1/1 Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/1 thiamine
HCI, 120.0 g/1 sucrose, 1.0 mg/12,4-D, and 2.88 g/1 L-proline (brought to
volume
with D-I H20 following adjustment to pH 5.8 with KOH); 2.0 g/1 Gelrite (added
after
bringing to volume with D-I H20); and 8.5 mg/1 silver nitrate (added after
sterilizing
the medium and cooling to room temperature). Selection medium (560R) comprises
4.0 g/1 N6 basal salts (SIGMA C-1416), 1.0 m1/1 Eriksson's Vitamin Mix (1000X
SIGMA-1511), 0.5 mg/1 thiamine HCI, 30.0 g/1 sucrose, and 2.0 mg/12,4-D
(brought
to volume with D-I H20 following adjustment to pH 5.8 with KOH); 3.0 g/1
Gelrite
(added aftex bringing to volume with D-I H20); and 0.85 mg/1 silver nitrate
and 3.0
mg/1 bialaphos(both added after sterilizing the medium and cooling to room
temperature).
Plant regeneration medium (288J) comprises 4.3 g/1 MS salts (GIBCO 11117-
074), 5.0 m1/1 MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/1
thiamine
HCL, 0.10 g/1 pyridoxine HCL, and 0.40 g/1 glycine brought to volume with
polished
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D-I H20) (Murashige and Skoog (1962) Physiol. Playzt. 15:473), 100 mg/1 myo-
inositol, 0.5 mg/1 zeatin, 60 gJl sucrose, and 1.0 m1/1 of 0.1 mM abscisic
acid (brought
to volume with polished D-I H20 after adjusting to pH 5.6); 3.0 g/1 Gelrite
(added
after bringing to volume with D-I H20); and 1.0 mg/1 indoleacetic acid and 3.0
mg/1
bialaphos (added after sterilizing the medium and cooling to 60°C).
Hormone-free
medium (272V) comprises 4.3 g/1 MS salts (GIBCO 11117-074), 5.0 m1/1 MS
vitamins stock solution (0.100 g/1 nicotinic acid, 0.02 g/1 thiamine HCL, 0.10
g/1
pyridoxine HCL, and 0.40 g!1 glycine brought to volume with polished D-I H20),
0.1
g/1 myo-inositol, and 40.0 g/1 sucrose (brought to volume with polished D-I
H20 after
adjusting pH to 5.6); and 6 g/1 bacto-agar (added after bringing to volume
with
polished D-I H20), sterilized and cooled to 60° C.
Particle Gun Treatment
The sample plates are bombarded at level #4 in particle gun #HE34-1 or
#HE34-2. All samples receive a single shot at 650 PSI, with a total of ten
aliquots
taken from each tube of prepared particles/DNA.
Subsequent Treatment
Following bombardment, the embryos axe kept on 560Y medium for 2 days,
then transferred to 5608 selection medium containing 3 mg/liter Bialaphos, and
subcultured every 2 weeks. After approximately 10 weeks of selection,
selection-
resistant callus clones are transferred to 288J medium to initiate plant
regeneration.
Following somatic embryo maturation (2-4 weeks), well-developed somatic
embryos
are transferred to medium for germination and transferred to the lighted
culture room.
Approximately 7-10 days later, developing plantlets are transferred to 272V
hormone-
free medium in tubes far 7-10 days until plantlets are well established.
Plants are then
transferred to inserts in flats (equivalent to 2.5" pot) containing potting
soil and grown
for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in
the
greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to
maturity.
Plants are monitored and scored for altered acyl-CoA thioesterase activity
and/or
altered oil content in kernels.
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EXAMPLE 3
Ag~obacteriunz-mediated Transformation of Maize
For Ag~obacter~ium-mediated transformation of maize with an acyl-CoA
thioesterase nucleotide sequence of the invention operably linked to a oleosin
promoter, preferably the method of Zhao is employed (U.S. Patent No.
5,981,840, and
PCT patent publication W098/32326; the contents of which are hereby
incorporated by
reference). Briefly, immature embryos axe isolated from maize and the embryos
contacted with a suspension of Agrobacterium, where the bacteria are capable
of
transferring the the acyl-CoA thioesterase nucleotide sequence to at least one
cell of at
least one of the immature embryos (step 1: the infection step). In this step
the
immature embryos are preferably immersed in an Ag~obacterium suspension for
the
initiation of inoculation. The embryos are co-cultured for a time with the
Ag~obacterium (step 2: the co-cultivation step). Preferably the immature
embryos are
cultured on solid medium following the infection step. Following this co-
cultivation
period an optional "resting" step is contemplated. In this resting step, the
embryos axe
incubated in the presence of at least one antibiotic known to inhibit the
growth of
Agrobacterium without the addition of a selective agent for plant
transformants (step
3: resting step). Preferably the immature embryos are cultured on solid medium
with
antibiotic, but without a selecting agent, for elimination of Agrobacterium
and for a
resting phase for the infected cells. Next, inoculated embryos are cultured on
medium
containing a selective agent and growing transformed callus is recovered (step
4: the
selection step). Preferably, the immature embryos are cultured on solid medium
with
a selective agent resulting in the selective growth of transformed cells. The
callus is
then regenerated into plants (step 5: the regeneration step), and preferably
calli grown
on selective medium are cultured on solid medium to regenerate the plants.
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EXAMPLE 4
Production of Transgenic Soybean by Embryo Transformation
Soybean embryos are bombarded with a plasmid containing an acyl-CoA
thioesterase nucleotide sequence of the invention operably linked to a oleosin
promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm in length
dissected from surface-sterilized, immature seeds of the soybean cultivar
A2872, are
cultured in the light or dark at 26°C on an appropriate agar medium for
six to ten
weeks. Somatic embryos producing secondary embryos are then excised and placed
into a suitable liquid medium. After repeated selection for clustexs of
somatic
embryos that multiplied as early, globular-staged embryos, the suspensions are
maintained as described below.
Soybean embryogenic suspension cultures can maintained in 35 ml liquid media
on a rotary shaker, 150 rpm, at 26°C with florescent lights on a 16:8
hour day/night
schedule. Cultures are subcultured every two weeks by inoculating
approximately
35 mg of tissue into 35 ml of liquid medium.
Soybean embryogenic suspension cultures may then be transformed by the
method of particle gun bombardment (I~lein et al. (1987) Nature (London)
327:70-73,
U.S. Patent No. 4,945,050). A Du Pont Biolistic PDS1000/HE instrument (helium
retrofit) can be used for these transformations.
A selectable marker gene that can be used to facilitate soybean transformation
is
a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell
et
al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from
plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188), and the 3'
region
of the nopaline synthase gene from the T-DNA of the Ti plasmid of
Agrobacte~ium
tumefaciens. The expression cassette comprising the acyl-CoA thioesterase
nucleotide sequence of the invention operably linked to a oleosin promoter can
be
isolated as a restriction fragment. This fragment can then be inserted into a
unique
restriction site of the vector carrying the marker gene.
To 50 ~1 of a 60 mg/ml 1 ~m gold particle suspension is added (in order): 5
~,l
DNA (1 ~,g/~,1), 20 ~.1 spermidine (0.1 M), and 50 ~l CaCl2 (2.5 M). The
particle
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preparation is then agitated for three minutes, spun in a microfuge for 10
seconds and
the supernatant removed. The DNA-coated particles are then washed once in 400
~,l
70% ethanol and resuspended in 40 ~,1 of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five microliters
of the
DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture is placed in
an empty 60x15 mm petri dish and the residual liquid removed from the tissue
with a
pipette. For each transformation experiment, approximately 5-10 plates of
tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi, and the
chamber
is evacuated to a vacuum of 28 inches mercury. The tissue is placed
approximately
3.5 inches away from the retaining screen and bombarded three times. Following
bombardment, the tissue can be divided in half and placed back into liquid and
cultured as described above.
Five to seven days post bombardment, the liquid media may be exchanged with
fresh media, and eleven to twelve days post-bombardment with fresh media
containing 50 mg/ml hygromycin. This selective media can be refreshed weekly.
Seven to eight weeks post-bombardment, green, transformed tissue may be
observed
growing from untransformed, necrotic embryogenic clusters. Isolated green
tissue is
removed and inoculated into individual flasks to generate new, clonally
propagated,
transformed embryogenic suspension cultures. Each new line may be treated as
an
independent transformation event. These suspensions can then be subcultured
and
maintained as clusters of immature embryos or regenerated into whole plants by
maturation and germination of individual somatic embryos.
EXAMPLE 5
Production of Transgenic Sunflower by Meristem Tissue Transformation
Sunflower meristem tissues are transformed with an expression cassette
containing an acyl-CoA thioesterase nucleotide sequence of the invention
operably
linked to a oleosin promoter as follows (see also European Patent Number EP 0
486233, herein incorporated by reference, and Malone-Schoneberg et al. (1994)
Plant
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Science 103:199-207). Mature sunflower seed (Helia~cthus annuus L.) are
dehulled
using a single wheat-head thresher. Seeds are surface sterilized for 30
minutes in a
20% Clorox bleach solution with the addition of two drops of Tween 20 per 50
ml of
solution. The seeds are rinsed twice with sterile distilled water.
Split embryonic axis explants are prepared by a modification of procedures
described by Schrammeijer et al. (Schrammeijer et al. (1990) Plant Cell Rep.
9: 55-
60). Seeds are imbibed in distilled water for 60 minutes following the surface
sterilization procedure. The cotyledons of each seed are then broken off,
producing a
clean fracture at the plane of the embryonic axis. Following excision of the
root tip,
the explants are bisected longitudinally between the primordial leaves. The
two
halves are placed, cut surface up, on GBA medium consisting of Murashige and
Skoog mineral elements (Murashige et al. (1962) Physiol. Plant., I5: 473-497),
Shepard's vitamin additions (Shepard (1980) in Emergent Techniques for the
Genetic
Improvement of Crops (University of Minnesota Press, St. Paul, Minnesota), 40
mg/1
adenine sulfate, 30 g/1 sucrose, 0.5 mg/16-benzyl-aminopurine (BAP), 0.25 mg/1
indole-3-acetic acid (IAA), 0.1 mg/1 gibberellic acid (GA3), pH 5.6, and 8 g/1
Phytagar.
The explants are subjected to microprojectile bombardment prior to
Ag~obacteriurn treatment (Bidney et al. (1992) Plant Mol. Biol. 18: 301-313).
Thirty
to forty explants are placed in a cixcle at the center of a 60 X 20 mm plate
for this
treatment. Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are
resuspended in 25 ml of sterile TE buffer (10 mM Tris HCI, 1 mM EDTA, pH 8.0)
and 1.5 ml aliquots are used per bombardment. Each plate is bombarded twice
through a 150 mm nytex screen placed 2 cm above the samples in a PDS 1000~
particle acceleration device.
Disarmed AgrobacteriunZ tumefaciehs strain EHA105 is used in all
transformation experiments. A binary plasmid vector comprising the expression
cassette that contains the acyl-CoA thioesterase nucleotide sequence of the
invention
operably linked to a oleosin promoter is introduced into Agrobacterium strain
EHA105 via freeze-thawing as described by Holsters et al. (1978) Mol. Gen.
Genet.
163:181-187. This plasmid further comprises a kanamycin selectable marker gene
(i.e, hptll). Bacteria for plant transformation experiments are grown
overnight (28°C
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and 100 RPM continuous agitation) in liquid YEP medium (10 gm/1 yeast extract,
10
gm/1 Bactopeptone, and 5 gm/1 NaCI, pH 7.0) with the appropriate antibiotics
required
for bacterial strain and binary plasmid maintenance. The suspension is used
when it
reaches an OD600 of about 0.4 to 0.8. The Agrobacte~ium cells are pelleted and
resuspended at a final OD600 of 0.5 in an inoculation medium comprised of 12.5
mM
MES pH 5.7, 1 gm/1 NH4Cl, and 0.3 gm/1 MgS04.
Freshly bombarded explants are placed in an Ag~obacte~ium suspension,
mixed, and left undisturbed for 30 minutes. The explants are then transferred
to GBA
medium and co-cultivated, cut surface down, at 26°C and 18-hour days.
After three
days of co-cultivation, the explants are transferred to 374B (GBA medium
lacking
growth regulators and a reduced sucrose level of 1%) supplemented with 250
mg/1
cefotaxime and 50 mg/1 kanamycin sulfate. The explants are cultured for two to
five
weeks on selection and then transferred to fresh 374B medium lacking kanamycin
for
one to two weeks of continued development. Explants with differentiating,
antibiotic-
resistant areas of growth that have not produced shoots suitable for excision
are
transferred to GBA medium containing 250 mg/1 cefotaxime for a second 3-day
phytohormone treatment. Leaf samples from green, kanamycin-resistant shoots
are
assayed for the presence of NPTII by ELISA and for the presence of transgene
expression by assaying for acyl-CoA thioesterase activity.
NPTII-positive shoots axe grafted to Pioneer~ hybrid 6440 ih vitro-grown
sunflower seedling rootstock. Surface sterilized seeds are germinated in 48-0
medium
(half strength Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH 5.6)
and
grown under conditions described for explant culture. The upper portion of the
seedling is removed, a 1 cm vertical slice is made in the hypocotyl, and the
transformed shoot inserted into the cut. The entire area is wrapped with
parafilm to
secure the shoot. Grafted plants can be transferred to soil following one week
of in
vitro culture. Grafts in soil axe maintained under high humidity conditions
followed
by a slow acclimatization to the greenhouse enviromnent. Transformed sectors
of TO
plants (parental generation) maturing in the greenhouse are identified by
NPTII
ELISA and/or by acyl-CoA thioesterase activity analysis of leaf extracts while
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transgenic seeds harvested from NPTII-positive TO plants are identified by
acyl-CoA
thioesterase activity analysis of small portions of dry seed cotyledon.
An alternative sunflower transformation protocol allows the recovery of
transgenic progeny without the use of chemical selection pressure. Seeds are
dehulled and surface-sterilized for 20 minutes in a 20% Clorox bleach solution
with
the addition of two to three drops of Tween 20 per 100 ml of solution, then
rinsed
three times with distilled water. Sterilized seeds are imbibed in the dark at
26°C for
20 hours on filter paper moistened with water. The cotyledons and root radical
are
removed, and the meristem explants are cultured on 374E (GBA medium consisting
of MS salts, Shepard vitamins, 40 mg/1 adenine sulfate, 3% sucrose, 0.5 mg/16-
BAP,
0.25 mg/1 IAA, 0.1 mg/1 GA, and 0.8% Phytagar at pH 5.6) for 24 hours under
the
dark. The primary leaves are removed to expose the apical meristem, around 40
explants are placed with the apical dome facing upward in a 2 cm circle in the
center
of 374M (GBA medium with 1.2% Phytagar), and then cultured on the medium for
24 hours in the dark.
Approximately 18.8 mg of 1.8 ~m tungsten particles are resuspended in 150 ~I
absolute ethanol. After sonication, 8 ~,l of it is dropped on the center of
the surface of
macrocarrier. Each plate is bombarded twice with 650 psi rupture discs in the
first
shelf at 26 mm of Hg helium gun vacuum.
The plasmid of interest is introduced into Agrobacterium tumefacievcs strain
EHA105 via freeze thawing as described previously. The pellet of overnight-
grown
bacteria at 28 °C in a liquid YEP medium (10 g/1 yeast extract, 10 g/1
Bactopeptone,
and 5 g/1 NaCI, pH 7.0) in the presence of 50 ~,g/1 kanamycin is resuspended
in an
inoculation medium (12.5 mM 2-mM 2-(N-morpholino) ethanesulfonic acid, MES, 1
g/1 NH4C1 and 0.3 g/1 MgS04 at pH 5.7) to reach a final concentration of 4.0
at OD
600. Particle-bombarded explants are transferred to GBA medium (374E), and a
droplet of bacteria suspension is placed directly onto the top of the
meristem. The
explants are co-cultivated on the medium for 4 days, after which the explants
are
transferred to 374C medium (GBA with 1% sucrose and no BAP, IAA, GA3 and
supplemented with 250 ~,g/ml cefotaxime). The plantlets are cultured on the
medium
for about two weeks under 16-hour day and 26 °C incubation conditions.
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Explants (around 2 cm long) from two weeks of culture in 374C medium are
screened for acyl-CoA thioesterase activity using assays known in the art [add
references to known assays if at all possible]. After positive (i.e., for
increased or
decreased acyl-CoA thioesterase activity expression) explants are identified,
those
shoots that fail to exhibit the desired acyl-CoA thioesterase activity are
discarded, and
every positive explant is subdivided into nodal explants. One nodal explant
contains
at least one potential node. The nodal segments are cultured on GBA medium for
three to four days to promote the formation of auxiliary buds from each node.
Then
they are transferred to 374C medium and allowed to develop for an additional
four
weeks. Developing buds are separated and cultured for an additional four weeks
on
374C medium. Pooled leaf samples from each newly recovered shoot are screened
again by the appropriate protein activity assay. At this time, the positive
shoots
recovered from a single node will generally have been enriched in the
transgenic
sector detected in the initial assay prior to nodal culture.
Recovered shoots positive for the desired acyl-CoA thioesterase expression
are grafted to Pioneer hybrid 6440 ih vitro-grown sunflower seedling
rootstock. The
rootstocks are prepared in the following manner. Seeds are dehulled and
surface-
sterilized for 20 minutes in a 20% Clorox bleach solution with the addition of
two to
three drops of Tween 20 per 100 ml of solution, and are rinsed three times
with
distilled water. The sterilized seeds are germinated on the filter moistened
with water
for three days, then they are transferred into 48 medium (half strength MS
salt, 0.5%
sucrose, 0.3 % gelrite pH 5.0) and grown at 26 °C under the dark for
three days, then
incubated at 16-hour-day culture conditions. The upper portion of selected
seedling
is removed, a vertical slice is made in each hypocotyl, and a transformed
shoot is
inserted into a V-cut. The cut area is wrapped with parafilm. After one week
of
culture on the medium, grafted plants are transferred to soil. In the first
two weeks,
they are maintained under high humidity conditions to acclimatize to a
greenhouse
environment.
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention
pertains. All
publications and patent applications are herein incorporated by reference to
the same
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extent as if each individual publication or patent application was
specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
obvious
that certain changes and modifications may be practiced within the scope of
the
appended claims.
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Zheng, Pei~hong
Nichols, Scott
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CA 02414408 2003-O1-03
WO 02/04648 PCT/USO1/21458
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CA 02414408 2003-O1-03
WO 02/04648 PCT/USO1/21458
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3
